Cloning and sequencing of the allergen DAC G 5 of dactylis glomerata pollen, its preparation and use thereof

ABSTRACT

A purified nucleic acid molecule comprising a nucleotide sequence coding for allergen Dac g  5  having amino acid sequence SEQ ID NO. 2, a derivative or a fragment thereof.

RELATED APPLICATION

This application is a divisional of application Ser. No. 10/303,426, filed Nov. 25, 2002, which is a continuation of PCT/FR01/01666 filed May 29, 2001, which claims priority of French Application No. 00/06857 filed May 29, 2000, incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the cloning and sequencing of Dactylis glomerata pollen allergens, more particularly, the allergen Dac g 5. The invention also relates to the production of the recombinant allergen for incorporation in preparations useful for the diagnosis or treatment of allergies.

BACKGROUND

Allergens are the most abundant proteins of pollen and constitute the major cause of allergies in temperate regions.

Certain genetically predisposed individuals become hypersensitive (allergic) to antigens stemming from extremely varied environmental sources. Antigens capable of inducing an immediate or delayed hypersensitization reaction are referred to as allergens. Allergens can have as their origin notably trees, herbaceous plants, insects, mammals, food, drugs and chemical products. Allergens are classified in groups I to V according to their immunochemical properties. The allergen Dac g 5 of Dactylis glomerata belongs to group V as does the allergen Lol pV of Lolium perenne.

The antibodies involved in allergy belong to the IgE class of immunoglobulins. In the presence of an allergen, IgE binds to mastocytes and basophils, which leads to the release by these cells of different chemical mediators and thus to the manifestation of allergy. Allergy can be manifested in different forms such as, e.g., anaphylactic shock, asthma, rhinitis or atopic dermatitis.

When the diagnosis of allergy to a particular compound has been established, desensitization of the patient in relation to the implicated allergen is the most frequent therapeutic approach, especially when the presence of the allergen cannot be avoided as in the case of pollen and acarids. This type of treatment has proven to be effective, but it requires the availability of an effective and safe product. In fact, the treatment presents a risk of anaphylactic shock such that the administered product must be free of any impurities that could constitute another potential allergen. At present, it is only known to use complex mixtures of allergens and not pure products. It is, therefore, necessary to have available allergens in a structural form as close as possible to the natural allergen and having the highest possible degree of purity.

One of the possible means for attaining this goal is the production of recombinant allergens in a host organism (Laffer, S. et al., J. Allergy Clin. Immunol., September 1996, volume 98, no. 3, pages 652-658).

As examples, we can cite the patent application published as No. 819 763 which describes the production of the modified allergen Der f II. The European patent published as No. 406 286 describes the cloning of a major allergen of rye grass pollen, Lol p 1, and the expression of this gene. The patent application published as No. 473 111 also discloses the production of recombinant acarid allergens used for desensitization. The patent application published as No. 463 059 pertains to allergens taken from ragweed and the use of these proteins. These applications disclose the expression of genes and the production of proteins in E. coli.

This system of expression has the disadvantage of not enabling the post-translational modifications of the proteins which can be implemented in eukaryote cells. For example, the proteins produced are not glycosylated. However, the glycosylation of certain allergen proteins can be important for their ability to bind to IgE (Van Ree et al., J. Biol. Chem., 2000, volume 275, pages 11451-11458).

SUMMARY OF THE INVENTION

This invention relates to a purified nucleic acid molecule including a nucleotide sequence coding for allergen Dac g 5 having amino acid sequence SEQ ID NO.2, a derivative or a fragment thereof.

This invention also relates to a process for producing recombinant protein Dac g 5, an isoform, a fragment or a functional or immunologic equivalent thereof, including culturing a prokaryote or eukaryote organism transformed by a nucleic acid molecule under conditions and for a sufficient length of time to enable expression of the protein, and isolating proteins produced from the transformed organisms.

This invention further relates to a pharmaceutical composition for treating or diagnosing an allergy, including a therapeutically effective amount of a protein or an antibody directed against the protein.

This invention still further relates to a process for detecting sensitivity manifested by an individual to pollen of Dactylis glomerata, including contacting a sample obtained from an individual with a protein or an antibody directed against the protein under conditions enabling formation of an antigen/antibody complex, and detecting presence of the complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an amino acid sequence of the proform of the allergen Dac g 5.

FIG. 2A is the amino acid sequence of the proform of the sense primer.

FIG. 2B is the amino acid sequence of the proform of the allergen Dac g 5 and the positioning of the primers on the sequence.

DETAILED DESCRIPTION

This invention pertains to the cloning, sequencing and preparation of a purified nucleic acid molecule comprising a nucleotide sequence coding for the protein constituting the allergen Dac g 5 or for a derivative thereof. The invention also pertains to insertion of this nucleic acid into an expression vector and production of the recombinant protein in a host organism or microorganism, in plant cells and plants or parts of plants, and preferably in tobacco plant cell suspensions and tobacco plants. This allergen is produced for the purpose of using it in diagnostics and immunotherapy.

The production of allergens in plant cells such as, e.g., tobacco plant cells, has the advantage of enabling production of glycosylated recombinant proteins. This production means has not been used to date for the production of allergens.

The studies performed in the framework of this invention concerned:

-   -   cloning the cDNA of Dac g 5,     -   insertion of the cloned nucleic acid molecule in a suitable         expression vector,     -   production of recombinant Dac g 5 in biological systems,     -   immunologic tests of the purified allergen.

Dac g 5 is a 26.5-kDa protein of 265 amino acids recognized by at least about 90% of subjects allergic to grass pollens.

Allergen Dac g 5 of Dactylis glomerata belongs to the group V allergens. Nucleotide sequences coding for allergens homologous to Dac g 5 have been described in the prior art. The inventors defined degenerated oligonucleotides from these sequences to specifically amplify using the RT-PCR technique a cDNA coding Dac g 5. A cDNA fragment of expected size was cloned from a total RNA population of Dactylis glomerata and then sequenced. This fragment was used to define specific primers for the RACE-PCR 5′ and 3′ protocols. These protocols enabled completion of the 5′ and 3′ ends of the fragment. The complete cDNA coding for Dac g 5 was sequenced after amplification of the 5′ and 3′ ends.

The sequences obtained enabled definition of new specific primers of Dac g 5. These primers were used to clone the cDNA of the proform and mature form of the allergen. The nucleotide sequences of the primers used for the PCR reactions were the following:

-   -   sense primer (proform), represented in FIG. 2A (primer C1) and         as SEQ ID NO. 9 in the attached sequence listing:     -   5′GGG TCT AGA ATG GCG GTC CAG AAG TAC ACC 3′     -   antisense primer used for cloning the proform, represented in         FIG. 2A (primer F1) and as SEQ ID NO. 10 in the attached         sequence listing:     -   5′GGG GAG CTC TCA GAC TTT GTA GCC ACC GGC 3′     -   sense primer (mature form), represented in FIG. 2A (primer F2)         and as SEQ ID NO. 11 in the attached sequence listing:     -   5′AAG CTC GAG AAA AGA GCC GAC GCC GGC TAC ACC 3′     -   antisense primer used for cloning the mature form, represented         in FIG. 2A (primer R2) and as SEQ ID NO. 12 in the attached         sequence listing:     -   5′GGG GGC GGC CGC TCA GAC TTT GTA GCC ACC GGC 3′     -   The cDNA clones were sequenced and the amino acid sequence         corresponding to each nucleotide sequence was determined.

The inventors were, thus, able to clone and sequence the nucleic acid coding for the allergen Dac g 5 of Dactylis glomerata pollen. The invention consequently pertains to a purified nucleic acid molecule constituted by or comprising a nucleotide sequence coding for the allergen Dac g 5, a derivative or a fragment thereof. The amino acid sequence of the proform of the allergen Dac g 5 is represented in FIG. 1 and as SEQ ID NO. 2 in the attached sequence listing. A fragment of this sequence delimited by the amino acids in positions 25 to 290 constitutes the mature Dac g 5 protein. The amino acid sequence of the mature form of the allergen Dac g 5 is represented in FIG. 1 and as SEQ ID NO. 4 in the attached sequence listing.

FIG. 1 gives the nucleotide and peptide sequence of the proform of the allergen Dac g 5 (isoform 1). The underlined sequence corresponds to the absent signal sequence of the mature protein. The codons and amino acids which are different in isoform 2 are in boxes. Table 1 below indicates the variations observed between isoforms 1 and 2.

TABLE 1 Amino acid position Isoform 1 Isoform 2 40 Thr (ACC) Ala (GCT) 51 Thr (ACG) Lys (AAG) 265 Val (GTT) Ala (GCT)

FIG. 2A represents the sequences of the primers (SEQ ID NOS 9, 11, 10, and 12, respectively, in order of appearance) and FIG. 2B (SEQ ID NOS 1-2) the sequence of the proform of the allergen Dac g 5 and the positioning of the primers on this sequence.

The term “derivative of the protein constituting the allergen Dac g 5” is understood to mean a protein whose amino sequence differs by the modification, suppression or addition of one or more amino acids, but is functionally and/or immunologically equivalent to Dac g 5.

Such modifications can result from the degeneration of the genetic code or modifications of the nucleotide acid sequence by any molecular biology technique. The expert in the field is able to determine among these sequences those which have functional and immunological properties identical or substantially identical to or close to those of Dac g 5, e.g., by means of an antibody. In this context, the inventors cloned two isoforms of Dac g 5. The amino acid sequences of these mature isoforms of the allergen Dac g 5 are represented as SEQ ID NO. 6 and SEQ ID NO. 8 in the attached sequence listing.

The invention thus also envisages the isoforms of the protein constituting the allergen Dac g 5 and has a homology of amino acid sequences greater than about 50%, preferably greater than about 70% and especially preferably greater than about 90% with the sequence represented as SEQ ID NO. 2 in the attachment. The invention envisages, more particularly, a protein, functional derivative and immunologically equivalent to Dac g 5 whose amino acid sequence is selected from among the sequences SEQ ID NO. 6 and SEQ ID NO. 8 in the attached sequence listing.

The term “fragment of the protein constituting the allergen Dac g 5” is understood to mean any peptide or polypeptide stemming from the protein, more particularly useful for the diagnosis of allergy.

A purified nucleic acid molecule comprising or constituted by a nucleotide sequence (cDNA) coding for the proform of the allergen Dac g 5 is represented as SEQ ID NO. 1 in the attached sequence listing. The invention also pertains to a derivative or fragment thereof and, more particularly, a nucleic acid molecule coding for the mature protein which is delimited by the nucleotides in positions 75 to 870 of the nucleotide sequence represented as SEQ ID NO. 1 in the attached sequence listing. This sequence is represented as SEQ ID NO. 3 in the attached sequence listing.

The invention also pertains to the nucleic acid molecules coding the isoforms of the mature Dac g 5 protein and, more particularly, those whose amino acid sequences are represented as SEQ ID NO. 6 and SEQ ID NO. 8 in the attached sequence listing.

On the basis of the nucleotide sequences coding for Dac g 5 or one of its isoforms, the expert in the field can define nucleotide sequences coding for proteins or polypeptides corresponding to a fragment of Dac g 5 or one of its isoforms and possessing, e.g., at least one epitope of Dac g 5 or one epitope of one of the isoforms of Dac g 5. The expert in the field can also define a nucleotide sequence coding for proteins functionally equivalent to Dac g 5 or to one of its isoforms but whose amino acid sequence is not identical to that of Dac g 5 or to that of one of its isoforms.

Finally, the expert in the field can define a nucleotide sequence coding for proteins immunologically equivalent to Dac g 5 or to one of its isoforms. These proteins are, e.g., capable of binding to the anti-Dac g 5 antibodies, but do not possess the enzymatic function of the natural Dac g 5 allergen. The phrase “derivative of a nucleic acid according to the invention” is understood more particularly to mean a nucleic acid molecule capable of hybridizing under standard hybridization conditions with one of the sequences represented as SEQ ID NO. 1 and SEQ ID NO. 3 in the attached sequence listing. These comprise, for example, the nucleotide sequences coding the isoforms of Dac g 5 represented as SEQ ID NO. 5 and SEQ ID NO. 7 in the attached sequence listing.

The invention also includes the mutagenesis of the protein enabling introduction at certain defined positions of the protein one or more sites carrying a particular functional group. Thus, one can introduce, e.g., an N-glycosylation site on the allergen.

The invention also pertains to a recombinant nucleic acid molecule comprising a polynucleotide sequence coding for the allergen Dac g 5 or one of its isoforms or a derivative thereof such as a fragment or a functional and/or immunologic equivalent of the protein Dac g 5 or one of its isoforms, a promoter bound in a functional manner to the sequence, possibly a selection gene placed under the control of its own promoter or of the same promoter as the sequence, and advantageously a termination sequence placed downstream of the sequence. It can be a cassette or preferably an expression vector comprising notably an origin of eukaryote or prokaryote replication, an adapted promoting sequence, a selection marker and a nucleotide sequence coding for the allergen Dac g 5 or one of its isoforms, or a derivative thereof placed under the control of said regulation sequences. The expert in the field can select without difficulty, from among the expression vectors known in the prior art the vector the best adapted to the host organism in which the protein is produced.

The invention envisages most particularly for the production of the protein constituting the allergen Dac g 5 or one of its isoforms a vector enabling expression of the nucleic acid in eukaryote cells, and preferably in plant cells or yeasts.

The expression vector can also be constructed in a manner to enable production of the previously defined recombinant protein in the form of a fusion protein. The polypeptide fused to the protein of interest can notably be useful to enable or facilitate its purification. This polypeptide can in particular be a sequence constituted of multiple histidines or histidine-tags added in a variable region not critical for the activity and conformation of the molecule, such as an internal region or a N-terminal or C-terminal end. The addition of a histidine-tag sequence enables purification of the recombinant protein by affinity chromatography on a chelated metal column.

The invention also pertains to an eukaryote or prokaryote host transformed by an expression vector as described above. This host can be, e.g., E. coli, Saccharomyces cerevisiae, Pichia pastoris or a plant cell, notably a Nicotiana tabacum cell in the genome of which is incorporated in a stable manner the nucleic acid molecule coding for the allergen Dac g 5 or one of its isoforms or a derivative thereof.

The invention also pertains to an organism or microorganism, preferably a cell or a plant, more preferably a tobacco plant cell which has incorporated in its genome, advantageously in a stable manner, a nucleic acid molecule of the invention placed under regulation sequence control in a manner to express the allergen Dac g 5 or one of its isoforms in these cells, in a plant or a determined part of the plant.

Transgenic plants according to the invention can be prepared by transforming a plant cell with the nucleic acid molecule then regenerating a plant from the transformed cell.

The invention pertains to a process for producing recombinant Dac g 5 protein or one of its isoforms, or a polypeptide fragment of Dac g 5 or one of its isoforms, or a functional or immunologic equivalent of Dac g 5 or one of its isoforms. This process comprises the culture of a prokaryote or eukaryote organism transformed by an expression vector as previously defined under conditions and over a sufficient length of time to enable expression of the protein.

This process also comprises isolation of the produced proteins from the culture of transformed organisms. In the particular case of the expression of recombinant proteins in tobacco plant cells, the cells expressing the allergen of interest are selected by immunodetection using an antibody directed against the natural form of Dac g 5. The allergens are localized by cell fractionation. Then they are purified from transgenic cell suspensions by immunodetection with an antibody directed against the natural form of Dac g 5. The process according to the invention also comprises the structural and immunologic analysis of the protein(s) produced.

The invention, thus, also pertains to the recombinant allergen Dac g 5 or a derivative thereof obtained by the process. The invention pertains to the proform and the mature form of the allergen Dac g 5. The invention also pertains to the isoforms of the mature form of Dac g 5. It also pertains to peptide or polypeptide fragments of Dac g 5 or of one of its isoforms, as well as proteins that are functionally or immunologically equivalent to Dac g 5 or to one of its isoforms. The proteins equivalent to Dac g 5 or to one of its isoforms can be obtained notably by directed mutagenesis applied to the DNA molecule coding for Dac g 5 or for one of its isoforms. The invention also pertains to a recombinant fusion protein comprising the protein Dac g 5 or one of its isoforms, a fragment of these proteins or a functional or immunologic equivalent.

This recombinant allergen, a derivative thereof, like the natural allergen, can be used for preparing monoclonal or polyclonal antibodies. The monoclonal antibodies are prepared according to the conventional techniques with which the expert in the field is quite familiar. The polyclonal antibodies can be obtained by immunizing animals with the allergen Dac g 5 by means of a suitable adjuvant; the antibodies are then purified from the serum of the immunized animals.

These antibodies can notably be employed to detect the presence of the allergen Dac g 5, a peptide fragment of the protein or one of its isoforms or an immunologic equivalent thereof, in a particular medium such as, e.g., a culture medium.

The invention furthermore pertains to pharmaceutical compositions intended for the treatment and/or diagnosis of an allergy and comprising as active principle an effective quantity of Dac g 5, a fragment of one of the isoforms of the protein or a functional or immunologic equivalent thereof, or an antibody directed against them. In these compositions, the active principle is combined with a pharmaceutically acceptable vehicle. The compositions intended for the treatment of allergy are formulated according to suitable principles known by the expert in the field such that it can be injected via the subcutaneous route or administered by any other route.

The invention also pertains to a process for the detection of sensitivity manifested by an individual to herbaceous pollen and, in particular, to the pollen of Dactylis glomerata. This process comprises the detection of the presence of antibodies binding to one of the isoforms of the recombinant Dac g 5 protein, a fragment or a functional and/or immunologic equivalent thereof, or a specific antibody of them. This process comprises notably a step during which a sample obtained from the individual is brought into contact with one of the isoforms of the recombinant Dac g 5 protein, a derivative of them or an antibody directed against them, under conditions enabling formation of an antigen/antibody complex, and then the detection of said complex.

The invention finally pertains to a reagent for diagnosis of an allergy characterized in that it comprises a preparation containing one of the isoforms of the recombinant protein Dac g 5 and/or a fragment and/or a derivative of one of the isoforms of Dac g 5, or an antibody directed against them. 

1. A recombinant protein comprising the allergen Dac g 5 or a derivative thereof obtained by culturing a prokaryote or eukaryote organism transformed by a nucleic acid molecule comprising a nucleotide sequence coding for allergen Dac g 5 having amino acid sequence SEQ ID NO. 2, a derivative or a fragment thereof, under conditions and for a sufficient length of time to enable expression of said protein, and isolating proteins produced from the transformed organisms.
 2. A recombinant protein comprising the allergen Dac g 5 or a derivative thereof obtained by culturing a prokaryote or eukaryote organism transformed by an expression vector comprising a purified nucleic acid molecule comprising a nucleotide sequence coding for allergen Dac g 5 having amino acid sequence SEQ ID NO. 2, a derivative or a fragment thereof, under conditions and for a sufficient length of time to enable expression of the protein, and isolating proteins produced from the transformed organisms.
 3. A pharmaceutical composition for treating or diagnosing an allergy, comprising a therapeutically effective amount of a protein according to claim 1 or an antibody directed against the protein.
 4. A reagent for diagnosing an allergy comprising a protein according to claim 1 or an antibody directed against the protein.
 5. A pharmaceutical composition for treating or diagnosing an allergy, comprising a therapeutically effective amount of a protein according to claim 2 or an antibody directed against the protein.
 6. A reagent for diagnosing an allergy comprising a protein according to claim 2 or an antibody directed against the protein.
 7. A process for detecting sensitivity manifested by an individual to pollen of Dactylis glomerata, comprising: contacting a sample obtained from an individual with a protein according to claim 1 or an antibody directed against the protein under conditions enabling formation of an antigen/antibody complex, and detecting presence of the complex. 